Bottom Line:
Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture.Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably.In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis.

ABSTRACTThe analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78-83%), high retention of cell viability (71-74%), high tumour cell enrichment against leukocytes (1.7-2 × 10(3)), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

f5: In vivo study of tumourigenicity of 4T1 and 4T07 CTCs in mouse models.(A): 4T07 and 4T1 viable CTCs captured by the bilayer microfilter device on-chip 21 days after their enrichment from whole blood, and their corresponding primary tumours in culture. In both tumour types, CTCs were found inside the central pore as well as around the central pore and in the margins of the bilayer membrane device, indicating their ability to migrate away from the original site capture. (B): Fluorescent intensities of primary tumour cells and viable CTCs cultured in the presence of 6-TG treatment (orange) compared to untreated controls (green). The fluorescent intensities of tumour cells with no TiterBlue reagent (blue) and 6-TG + culture media alone (purple) were also measured as a control, and demonstrate that there was no auto fluorescence contributing to the signals detected in treated versus untreated samples. (AU = Arbitrary Units). (C): H&E staining of FFPE sections from viable 4T1 and 4T07 CTCs captured by the SB microfilter. CTCs injected into mammary fads of mice demonstrate the formation of poorly differentiated, invasive tumours characteristically similar to their corresponding parental cell lines.

Mentions:
Primary tumours were established in 100% (6/6) of mice injected, and in vitro cell cultures from each primary tumour digested were successfully established in all cases. Cell cultures were established from blood samples in 33% (1/3) of mice injected with 4T1 and 4T07 cells, respectively, demonstrating SB microfilter's ability to permit tumour cell expansion directly on-chip (Figure 5A). An advantage of this in vivo mouse model system is that 4T1 and 4T07 tumour cells are resistant to treatment with 6-Thioguanine (6-TG)40. Thus, to confirm whether cells captured by the SB microfilter device originated from their primary tumours, viable cells were mechanically released by physical separation of the SB microfilter layers, placed in 96-well plates in triplicate, and grown in the presence of RPMI culture media supplemented with 50 mM 6-TG (Sigma-Aldrich, St Louis MO) for 5 days. After drug treatment, cell viability was assessed using the CellTiter Blue assay (Promega, Madison WI) where viable cells reduce resazurin to resourufin, which produces a fluorescent signal that can be detected by a plate reader at 590 nm. In contrast to SKBR-3 breast cancer cells that demonstrate sensitivity to 6-TG treatment, primary tumours and corresponding CTCs from 4T1 and 4T07 cells show similar viability in the presence of 6-TG compared to untreated controls (Figure 5B). Further, the execution of this experiment demonstrates the ability to release cells from the SB microfilter with efficiency and successfully culture tumour cells on surfaces other than the parylene-C membrane.

f5: In vivo study of tumourigenicity of 4T1 and 4T07 CTCs in mouse models.(A): 4T07 and 4T1 viable CTCs captured by the bilayer microfilter device on-chip 21 days after their enrichment from whole blood, and their corresponding primary tumours in culture. In both tumour types, CTCs were found inside the central pore as well as around the central pore and in the margins of the bilayer membrane device, indicating their ability to migrate away from the original site capture. (B): Fluorescent intensities of primary tumour cells and viable CTCs cultured in the presence of 6-TG treatment (orange) compared to untreated controls (green). The fluorescent intensities of tumour cells with no TiterBlue reagent (blue) and 6-TG + culture media alone (purple) were also measured as a control, and demonstrate that there was no auto fluorescence contributing to the signals detected in treated versus untreated samples. (AU = Arbitrary Units). (C): H&E staining of FFPE sections from viable 4T1 and 4T07 CTCs captured by the SB microfilter. CTCs injected into mammary fads of mice demonstrate the formation of poorly differentiated, invasive tumours characteristically similar to their corresponding parental cell lines.

Mentions:
Primary tumours were established in 100% (6/6) of mice injected, and in vitro cell cultures from each primary tumour digested were successfully established in all cases. Cell cultures were established from blood samples in 33% (1/3) of mice injected with 4T1 and 4T07 cells, respectively, demonstrating SB microfilter's ability to permit tumour cell expansion directly on-chip (Figure 5A). An advantage of this in vivo mouse model system is that 4T1 and 4T07 tumour cells are resistant to treatment with 6-Thioguanine (6-TG)40. Thus, to confirm whether cells captured by the SB microfilter device originated from their primary tumours, viable cells were mechanically released by physical separation of the SB microfilter layers, placed in 96-well plates in triplicate, and grown in the presence of RPMI culture media supplemented with 50 mM 6-TG (Sigma-Aldrich, St Louis MO) for 5 days. After drug treatment, cell viability was assessed using the CellTiter Blue assay (Promega, Madison WI) where viable cells reduce resazurin to resourufin, which produces a fluorescent signal that can be detected by a plate reader at 590 nm. In contrast to SKBR-3 breast cancer cells that demonstrate sensitivity to 6-TG treatment, primary tumours and corresponding CTCs from 4T1 and 4T07 cells show similar viability in the presence of 6-TG compared to untreated controls (Figure 5B). Further, the execution of this experiment demonstrates the ability to release cells from the SB microfilter with efficiency and successfully culture tumour cells on surfaces other than the parylene-C membrane.

Bottom Line:
Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture.Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably.In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis.

ABSTRACTThe analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78-83%), high retention of cell viability (71-74%), high tumour cell enrichment against leukocytes (1.7-2 × 10(3)), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.